GTP-binding proteins (“GTPases”) are a family of regulatory proteins that act as molecular switches. GTPases control a wide range of biological processes including: receptor signaling, intracellular signal transduction pathways, and protein synthesis. Their activity is regulated by factors that control their ability to bind GTP and hydrolyze it to GDP.
A group of GTPase proteins, named for their association with rat sarcomas (ras), was originally isolated from Harvey (H-ras, Ha-ras, rasH) and Kirsten (K-ras, Ki-ras, rasK) murine sarcoma viruses. Ras genes are widely conserved among animal species, and sequences corresponding to both H-ras and K-ras genes have been detected in human, avian, murine, and non-vertebrate genomes. The closely related N-ras gene was originally detected in human neuroblastoma and sarcoma cell lines. All genes of the family have a similar exon-intron structure and each encodes a p21 protein.
Although mammalian genomes contain three ras genes, mutations in kras are most frequently associated with human cancer (Bos, “Ras Oncogenes in Human Cancer: A Review,” Cancer Res. 49:4682-4689 (1989)). Therefore, properties that are specific to K-Ras are of particular significance to cancer biologists since they might be exploited in the development of anti-cancer drugs. The differential biology of Ras isoforms is generated, in large part, by distinct membrane targeting sequences. Membrane association of all Ras isoforms requires prenylation (i.e., farnesylation), proteolysis, and carboxyl methylation of a C-terminal CAAX motif. Plasma membrane targeting of the principal splice variant of K-Ras also requires a unique polybasic region adjacent to the CAAX motif (Hancock et al., “A Polybasic Domain or Palmitoylation is Required in Addition to the CAAX Motif to Localize p21ras to the Plasma Membrane,” Cell 63:133-139 (1990); Jackson et al., “Polylysine Domain of K-Ras 4B Protein is Crucial for Malignant Transformation,” Proc. Natl. Acad. Sci. USA 91:12730-12734 (1994); Choy et al., “Endomembrane Trafficking of Ras: The CAAX Motif Targets Proteins to the ER and Golgi,” Cell 98:69-80 (1999)).
K-Ras thus falls into a broad class of proteins that are anchored to the cytoplasmic face of the plasma membrane by virtue of post-translational modification with lipids that act in conjunction with polybasic stretches of polypeptide. Whereas the lipid moieties are thought to insert into the phospholipid bilayer, the polybasic regions are believed to associate with the anionic head groups of inner leaflet phospholipids (Leventis et al., “Lipid-Binding Characteristics of the Polybasic Carboxy-Terminal Sequence of K-Ras4B,” Biochemistry 37:7640-7648 (1998)). Included in this class of proteins is the myristoylated alanine-rich C kinase substrate (“MARCKS”) that associates with the plasma membrane via an N-terminal myristoyl modification and a polybasic region. Serine residues within the polybasic region are sites for PKC-mediated phosphorylation (FIG. 1A) that neutralize the charge and thereby cause the MARCKS protein to dissociate from the plasma membrane. The mechanism by which MARCKS is discharged from the plasma membrane through phosphorylation has been referred to as a myristoyl-electrostatic switch (McLaughlin et al., “The Myristoyl-Electrostatic Switch: A Modulator of Reversible Protein-Membrane Interactions,” Trends Biochem. Sci. 20:272-276 (1995)).
Like MARCKS, the polybasic region of K-Ras harbors three potential phosphorylation sites and this segment has previously been shown to be phosphorylated by PKC (Ballester et al., “Phorbol Ester- and Protein Kinase C-Mediated Phosphorylation of the Cellular Kirsten Ras Gene Product,” J. Biol. Chem. 262:2688-2695 (1987)). K-Ras was therefore tested for an electrostatic switch analogous to that of MARCKS (McLaughlin et al., “The Myristoyl-Electrostatic Switch: A Modulator of Reversible Protein-Membrane Interactions,” Trends Biochem. Sci. 20:272-276 (1995)).
The present invention is directed to determining whether K-Ras has a prenyl-electrostatic switch and, if so, the use of such a switch.